Synthetic antisense oligonucleotides have been used to inhibit DNA replication and protein synthesis with very high specificity(1,2). Various inhibition mechanisms have been proposed including:
1. Prevention of ribosomal complex assembly or mRNA translation (by hybridization of the antisense oligonucleotide to its target RNA molecule).
2. Degradation of the resultant DNA/RNA duplex by RNaseH.
3. Inhibition of the pre-mRNA splicing.
4. Formation of triple helix DNA structures.
Recent advances in antisense technology have been focused on modifying oligonucleotides in order to offer improved nuclease resistance and increased binding affinity. These approaches include: (a) Backbone modification (b) Sugar modification, and (c) Base modification The first generation of antisense oligonucleotides was based on backbone modification in which the backbone phosphodiester bond was replaced by (a) phosphorothioates, (b) phosphorodithioates, (c) methylphosphonates, (d) phosphotriesters and (e) phosphoramidates.
The phosphorothioate(3) analogues have some potential advantages since they (i) form relatively stable duplexes with RNA (−0.1° to −1° per modification); (ii) activate RNaseH degradation; (iii) are stable to cleavage by nucleases, and (iv) are stable to base catalyzed hydrolysis. Phosphorodithioates are also quite resistant to nuclease activity however they have little advantages over phosphorothioate derivatives for antisense applications(5). Like the phosphorothioates, methylphosphonates are normally obtained as mixtures. Although methylphosphonates do not activate RNaseH(4), they are uncharged and display (i) increased hydrophobicity, (ii) increased cell membrane permeability and (c) nuclease resistance. Regarding the O-alkylphosphotriesters(O-Et)(6), these oligomers strongly hybridize to RNA and closely conform to the helical conformation of natural β-phosphodiester DNA (self-complementary duplexes are substantially less stable(7)). The use of the latter molecules (phosphonates and triesters) in cell culture systems is limited due to the following drawbacks: (i) their low aqueous solubility; (ii) their reduced hybridization property due to high numbers of diastereoisomers formed by the chiral phosphorus atoms (phosphonates); (iii) due to their enhanced lipophilicity they are presumably targeted to intracellular lipid particles and membranes, and (iv) they are sensitive to base catalyzed hydrolysis. Phosphoramidates are quite resistant to nucleases but exhibit rather poor hybridization characteristics with DNA. This is not the case with 3′-NH phosphoramidates where substantial increase in Tm was observed.
An attractive approach in the development of antisense agents for DNA and RNA recognition is the polyamide (also known as peptide) nucleic acid (PNA) surrogates. PNAs are the first successful substitute for the sugar-phosphate backbone that have displayed equal or better binding affinity than natural DNA or RNA(8). In contrast to the various backbone units, PNAs do not bear any structural resemblance to natural oligonucleotides. PNAs bind to an oligonucleotide sequence either via a parallel mode where the PNA amino terminus is aligned with the 5′ end of DNA or via an anti parallel mode (aligned with the 3′ end). Hybridization through the antiparallel mode was found to be significantly more stable than the corresponding parallel hybrid and impart an extra Tm stability of 1.45°/modification and 1°-1.2°/modification for PNA-RNA and PNA-DNA duplexes, respectively. The alternative parallel binding mode is still as stable as DNA-RNA or DNA-DNA duplexes, formed by displacing the homopyrimidine DNA stretch from the DNA duplex(9).
Transcription inhibition by PNAs can occur either by triple helix formation or by strand displacement in which the PNA displaces one DNA strand in the DNA duplex to form a PNA-DNA hybrid. Following this, by binding to a further PNA oligomer, a local (PNA)2-DNA triple helix can be formed for certain sequences. Both PNA strands must be oriented either parallel or antiparallel to the DNA strand.
Two pivotal obstacles are implicated with the application of PNA systems: i) Low solubility, and ii) Diminished cell uptake. In this context and in order to cope with these hurdles several modifications yielding new types of polyamide building blocks were introduced such as depicted in the following formulae:

Compound A represents the original structure (Nielsen) of a PNA unit where the backbone part is composed of N-(2-aminoethyl) glycine chain and the nucleobase is tethered through an amide bond to the inner amino group. Such a highly hydrophobic system is of course of low solubility and consequently of diminished cell permeation ability. Compounds B and C and D represents a new polyamide backbone where the carboxylic moiety is replaced by phosphono (B,C) and phosphoro (D) groups to attain a more hydrophilic ribbon cord with a resistant to nucleases degradation(10). In contrast to B, compounds C and D consist of a delta hydroxy acid backbone. This allows chain elongation by methods adopted in solid phase synthesis of oligonucleotide. Compound E (OPNA) which consist an delta amino acid was designed as an ether analogue to afford the main chain sufficient flexibility and an improved water solubility(10). On the other hand the presence of a chiral center in the backbone structure extend chemical diversity. Structures F—H are true peptide nucleic acids analogues bearing nucleobases linked through an ethylene chain to C-1 and the amino group N of glycine and to C-4 (Trans) of proline in F,G,H respectively(11). One of the drawbacks of a polypeptide chain as a carrier of nucleobases is ascribed to its augmented rigidity, which interrupts the spatial hybridization properties of the PNA. Compound I is a chiral Delta-amino acid PNA with a partly conformationally constrained backbone derived of cyclohexyl moiety(12).
One particular PNA analog in which the linkage of the a nucleobase to the interior amino group of the PNA unit is via an ethylene bridge has previously been described for thymine(3). The synthesis of this derivative was accomplished from protected N,N-Bis-2-ethylamino glycine and acylisocyanate, which does not afford a general method of synthesizing other units. More importantly, it was demonstrated in that study that the positively charged PNA analog had inferior properties compared to regular PNA units (reviewed by Falkiewicz, 1999, Ref. 14). This disclosure teaches away from the use of positively charged PNA analogs altogether.